The long term goals of this project are to investigate the mechanisms of intracellular Ph (Ph(i)) regulation and the modulation of those mechanisms by physiologically important extracellular perturbations in vascular smooth muscle cells (VSM). Regulation of Ph(i) VSM may play important roles in both normal VSM function and in common pathophysiological processes. The contractile state of VSM is integral to the normal control of blood pressure and regional distribution of blood flow to various organ systems. VSM contractility has been suggested to be altered by changes in pH(i). As well, VSM are known to be important targets of extracellular acid-base disturbances in vivo. pH(i) could be involved in the pathogenesis of two important cardiovascular diseases, atherosclerosis and hypertension. Inappropriate VSM proliferation is part of the atherogenic process leading to atherosclerosis. In VSM (and other cell types), mitogens have been demonstrated to modulate the regulation of pH(i) by changing the activity of acid-base transporters. In addition, vasoactive agents such as angiotensin II (AngII) cause cell hypertrophy and polyploidy in VSM, morphological changes which also occur in VSM of the arterial wall during hypertension. In VSM (and other cell types), AngII has been shown to modulate pH(i) regulation by altering the activity of acid-base transporters. Defining the mechanisms of pH(i) regulation in VSM would also be important as a general model for mammalian intracellular pH regulation. To date, how pH(i) is determined by the activity of all the major cell- surface acid-base transporters has not been demonstrated in any mammalian cell except red blood cells. I propose to make the VSM a model system by (i) identifying three major acid-base transporters of these cells, (ii) determining how each transporter's activity is affected by pH(i) and (iii) using the information gained in (ii) to reconstruct the unperturbed steady-state condition, and the pH(i) response to perturbations. The latter involves reconstructing the kinetics of pH(i) recoveries from intracellular acid or alkali loads using the determined pH(i)-dependence of each transporter's activity. pH(i) will be measured in single VSM isolated from calf renal artery using the pH-sensitive probe BCECF and a microscope-based fluorimetric system. Transporter activity will be isolated pharmacologically using specific inhibitors. The second goal of this study is to understand how various factors which affect VSM function in vivo (so called "local metabolic factors") could modulate the regulation of pH(i). In other muscle cell types (skeletal and cardiac), contractile filament sensitivity to (changes in) Ca++ is dependent on pH(i). If this were also true for VSM, then changes in vascular tone could be due to changes in pH(i) as well as (or instead of) calcium ion. Some local metabolic factors have been shown to alter pH(i) regulation in other cell types, including changes in extracellular acid/base status, osmolarity, K+, lactate, and exposure to mitogens or vasoactive agents. I propose to study the effect of these factors on the pH(i)-dependence of each of the three major acid-base transporters. The transient and steady-state response of pH(i) to acute exposure to these factors will then be explained on the basis of the factors' effects on each of the three individual transport systems.